Recently, there has been a growing interest in microfluidic flow cytometry. Several chip based systems have been demonstrated for cytometry and sorting of cells and molecules. See, for example, Unger et al., Science, 2000, 288, 113-116; Fu et al., Nature Biotechnol., 1999,17, 1109-1111; Quake et al., Science, 2000, 290, 1536-1540; Schrum et al., Anal. Chem., 1999, 71, 4173-4177; Knight et al., Phys Rev. Lett., 1998, 80, 3863-3866; Chou et al., Proc. Natl. Acad. Sci. USA, 1999, 96, 11-13; Chou et al., Electrophoresis, 2000, 21, 81-90; and Kameoka, et al., Sensors and Actuators B, 2001, 77, 632-637. There are generally two ways to pump fluid in these devices: pressure driven flow or by electroosmotic forces. Pressure driven flow results in Poiseulle flow, which has a parabolic velocity distribution in the channel. This complicates measurement of analytes since each analyte, e.g., cell or molecule, passes through the interrogation region with a different velocity. One can mitigate the effects of the Poiseulle flow by using a sheath fluid for hydrodynamic focusing, but this introduces other issues by diluting the sample and complicating downstream analysis. Although electroosmotic flow is typically more uniform and plug-like than pressure driven flow, it too results in variability in flow velocity. See, for example, Schrum et al., Anal. Chem., 1999, 71, 4173-4177. In addition, most, if not all, electroosmostic flow requires careful balancing of the ions in the solution and attention to prevent ion depletion. Furthermore, in some cases, it has also been shown that eukaryotic cells are difficult to manipulate electroosmotically. See, for example, Li et al., Anal. Chem., 1997, 69, 1564-1568.
One possible method for measuring a velocity independent characteristic parameter, e.g., fluorescence, of an analyte is to use a uniform detection zone, e.g., excitation region, large enough to illuminate the entire particle or molecule of interest. In this case, the height of the detected fluorescent peak will be substantially proportional to the fluorescence intensity of the particle. See, for example, Chou et al., Proc. Natl. Acad. Sci. USA, 1999, 96, 11-13. While this method is substantially not affected by the distribution of velocities, using only one point from the entire peak, namely its maximum, exploits only a small part of the information that is embedded in the peak. Moreover, the accuracy of this method is susceptible to noises. On the other hand, measuring the area underneath the entire peak, which is proportional to total fluorescence intensity integrated over the excitation duration, is velocity dependent measurement. This is due to the fact that faster particles have narrower peaks than slower particles. This will result in integrals being inversely proportional to the velocity of the particle. One solution is to normalize the area of each peak by the velocity of the corresponding particle to obtain velocity independent measurement of the fluorescence intensity.
Several methods for measuring the velocity in microfluidic devices have been reported. In particle image velocimetry, video imaging is used to measure the velocities of particles in a channel by observing the displacement of the particles within a known time interval. See, for example, Singh et al., Anal. Chem., 2001, 73, 1057-1061; Barker et al., Anal. Chem., 2000, 72, 5925-5929; and Santiago et al., Experiments in Fluids, 1998, 25, 316-319. This method is advantageous in obtaining the velocity spatial distribution, however it is not suitable for accurately measuring other analyte characteristic parameters, such as the fluorescent intensity.
Shah convolution Fourier transform is another method to measure velocity in microfluidic devices. See, for example, Kwok et al., Anal. Chem., 2001, 73, 1748-1753 and Crabtree et al., Anal. Chem., 1999, 71, 2130-2138. In this method, a mask with a periodic array of slits spatially modulates the excitation beam. When an analyte is moving in the beam the spatial modulation is converted into a temporal modulation. The distribution of velocities is found by Fourier transforming the temporal signal and identifying the peaks. However, such a practical use of this method has not been demonstrated. Moreover, the practical implementation of this method most likely will require fabrication of the mask on the chip which adds to the complexity of the device.
Therefore, there is a need for apparatuses and methods for determining a velocity independent analyte characteristic parameter.